Although a role of cAMP in the function of the male gamete is widely accepted, the exact mechanisms of cAMP generation during the maturation of the spermatozoon and at fertilization are largely unknown. Our laboratory has used both biochemical and genetic approaches to investigate the components of this pathway, as well as its role in sperm motility. We determined that at least two adenylyl cyclases, a membrane-bound AC3 and soluble adenylyl cyclases (sAC), contribute to cAMP production in spermatids and in spermatozoa. The properties of sAC have been extensively investigated demonstrating that this cyclase is integrated in positive and negative feedback loops regulating cAMP levels. Ablation of sAC in mice produces complete male infertility and major disruption in sperm motility. Inactivation of the membrane-bound ACS also causes a decrease in male fertility and impairs sperm functions. We propose to use these genetic in vivo models to further define the properties of cAMP signaling in the maturing spermatozoon, and their role in the control of motility, acrosome reaction, and during fertilization. The experimental plan is organized along three Specific Aims. The first Specific Aim will be devoted to further characterization of the sAC null phenotype, and the properties of cAMP signaling in spermatozoa devoid of sAC and AC3, including the mechanism of GPCR stimulation of motility. The second Specific Aim will focus on the biochemical properties of sAC and on the regulatory feedback controlling its activity. The last Specific Aim will investigate the relationship between cyclases and downstream targets in the spermatozoon. The existence of macromolecular complexes organizing cyclases, phosphodiesterases, and protein kinases will be investigated and their role in sperm function will be determined. These studies will offer insight into the regulation of processes essential for fertilization. They will also provide the groundwork for validation of targets for pharmacological manipulation of the male gamete, and new diagnostic tools useful to define the male factor.